1. Field of the Invention
The invention is directed toward biochemical assays, more particularly toward assays using fluorescence polarization detection with two or more fluorescent labels in the experiment.
2. Description of the Related Art
Fluorescence polarization (FP) assays are becoming popular, since they are homogeneous and relatively safe, with no radioactive material. A good discussion is provided in the recent review article by John Owicki entitled xe2x80x9cFluorescence Polarization and Anisotropy in High Throughput Screening: Perspectives and Primerxe2x80x9d, published in the Journal of Biomolecular Screening, Volume 5, No. 5, pp 297-306 (2000).
The technique has at its core the detection of relative intensity of fluorescence emission in two orthogonal states of polarization. The labels are probe molecules (probes) which are excited with linearly polarized light and, depending on the molecular rotation rate and the excitation lifetime, their fluorescence emission is preferentially polarized along the axis of the excitation beam to a greater or lesser extent. If the molecular rotation time is long compared with the excited-state lifetime, the polarization of the emission is more highly polarized; if the rotation time is short, the emission is more nearly random in polarization. Since chemical binding or other reactions alter the molecular rotation time, they alter the FP value and so can be detected.
FP is defined by the equation
Pxe2x89xa1[I81 xe2x88x92I195 ]/[I∥
+Ixe2x8axa5]=[I81 /Ixe2x8axa5xe2x88x921]/ 
[I81 /Ixe2x8axa5+1]xe2x80x83xe2x80x83[1]
where I∥ and I195  are the intensities of fluorescence emission polarized in the same sense as the polarization light and polarized orthogonal to it, respectively. There is a related concept termed fluorescence anisotropy (FA), which normalizes according to total fluorescence emission I=I∥+2I└ and is defined by the equation
rxe2x89xa1[I∥xe2x88x92Ixe2x8axa5]/[I81 
+2Ixe2x8axa5]=[I
∥/Ixe2x88x921]/[I∥/I∥
/I+2]xe2x80x83xe2x80x83[2]
One can convert between P and r using the equations
P=3r/(2+r)xe2x80x83xe2x80x83[3]
r=2P/(3xe2x88x92P)xe2x80x83xe2x80x83[4]
and in general, instrumentation or assays that provide a measurement of P will provide a measurement of r as shown in equations [3] and [4]; and vice versa. Similarly, instrumentation that provides an improved ability to measure one, will also provide an improved ability to measure the other. For simplicity, this specification refers to FP throughout, but is equally applicable to FA.
Measurements of FP are complicated by the presence of contaminant signals such as background fluorescence. These contribute fluorescence emissions with uncontrolled FP, shifting the measured FP. Optical filtering and other aspects of instrumental design are designed to minimize these signals. As reported by Owicki, post-processing by ratiometric corrections is a suitable way to correct these contaminants, since the contaminant is additive, rather than multiplicative, in nature.
There is at present no system or method for measuring an FP assay with multiple probes.
It is an object of the present invention to provide a system and method for performing multiple probe assays. It is another object to enable SNP (single nucleotide polymorphism) detection and coexpression using FP methods.
It is a further object of the invention is to measure the fluorescence polarization of two or more probes using the instrument described in my pending application Ser. No. 09/395,661 entitled xe2x80x9cFluorescence Polarization Assay System and Methodxe2x80x9d, the contents of which are hereby incorporated by reference, together with suitable optical filters for the probes involved; and to attain the high accuracy and self-calibration feature described therein for multiple probes.
Yet another object of the invention is to provide a method for measuring the fluorescence polarization of two or more probes using instruments of the prior art such as the LJL Analyst or Acquest.
Another aim of the invention is to provide methods for measuring the fluorescence polarization of two or more probes at once with high accuracy, utilizing instruments of the type described in my pending co-filed application entitled xe2x80x9cInstantaneous Dual Band Fluorescence Detection Systemsxe2x80x9d, application Ser. No. 09/793853, the contents of which are hereby incorporated by reference.
It is also a goal of the present invention to provide for self-calibration that yields accurate values of FP without need for a priori knowledge of the target FP values of the probes.
The invention resides in a system and method for measuring FP or FA for two or more probes in a single sample, even though they may have overlapping excitation spectra or emission spectra, or both.
The probes can be simultaneously excited by a single source, if desired. The instrument provides apparatus to separate the fluorescence emission flux according to its spectral band and quantify it. For example, this can consist of a filter wheel containing filters that preferentially transmit emission flux from each probe in turn; or a birefringent network and a double-refraction element that spatially separates light according to its wavelength band and captures multiple bands instantaneously. Other arrangements can also be used to achieve the goal of quantifying the flux in various emission bands and polarization states.
Alternatively, the probes can be excited separately through use of sequential excitation at various wavelength bands in turn. The fluorescence emission flux and polarization state is measured for each type of excitation.
The equipment and method are analogous whether the probes are differentially excited through choice of excitation band; or emit differentially into various emission bands. For simplicity a common nomenclature is used; in either case, a given spectral band is said to correspond to a given probe, whether it is an excitation band used to preferentially excite that probe, or an emission band in which that probe preferentially emits.
To measure N probes, a total of at least 2N pieces of data, and preferably 4N pieces of data are required, comprising the various combinations of excitation polarization state, emission polarization state, and spectral band. These measurements are the raw data from which one will calculate the FP of each probe. However, if one were to take the values obtained at the spectral band corresponding to a given probe, and plug them into the FP equations of the prior art, one would not obtain the desired result, namely an accurate value of FP for that probe. hen 4N pieces of data are taken, the measurement can be inherently self-calibrating, with no need for a priori knowledge about the FP properties of the probes being measured. Or, one may take a single full data set comprising 4N pieces of data, from which an instrumental calibration is derived; subsequent readings taken with a smaller set of 2N pieces of data can be processed to yield accurately calibrated values of FP. The process for taking an initial full set of measurements, deriving an instrumental calibration, then working with subsequent smaller sets of measurements to yield absolutely calibrated readings of FP/FA, is described in my pending co-filed application entitled xe2x80x9cAutomatic G-Factor Calibrationxe2x80x9d application Ser. No. 09/793856, which is hereby incorporated by reference.
The present invention provides, among other things, a method for determining an accurate value of FP for each probe from the various raw data measurements, in a way that correctly accounts for the complex multi-probe assay system, the cross-talk between probes, and the physical limitations of the instrument.
One can speak of cross-talk between probes, meaning the degree to which a given probe is detected when the instrument is seeking to measure a different probe (the target probe). This occurs because the probes fluoresce over broad wavelength ranges which overlap, even when they fluoresce most intensely in mutually exclusive ranges.
Mathematically, we write the instrument""s response to probe k when set to read probe j as ajk, and one can write the instrumental response function for all probes and instrumental settings as a matrix A populated with elements ajk. In such a matrix, the diagonal represents the response of the instrument to the target probes, while off-diagonal members represent cross-talk. For this reason, the A matrix is also called the cross-talk matrix for the probes and instrument involved. For two probes and two corresponding wavebands, this is a 2xc3x972 matrix, and the cross-talk is represented by the second diagonal. The degree of isolation between flux from the different probes is never perfect due to instrumental limitations. In the usual case where probes have partially overlapping spectra the separation is fundamentally limited by the spectral cross-talk between the probes.
The core of the invention is the quantification of FP for multiple probes through measurement of the cross-talk by means of the instrumental response matrix A, or an equivalent formalism, and the use of this cross-talk information to determine the contribution of each probe to the measured flux readings in each spectral band and polarization state. From these derived quantities, termed the probe contributions, one can accurately calculate the FP or FA for each of the probes.
The matrix A can be measured using control samples that contain only a single probe each. Indeed it is possible to characterize a set of four matrices {ahh, Ahv, Avh, Avv} for all possible excitation and emission polarization states, when the instrumental response is known to vary as a function of polarization. This can be caused by factors such as polarization-dependent transmission in dichroic mirror elements, which shifts the spectral response and thus alters the contents of matrix A. The set of matrices may then be used to derive accurate values of the probe contributions, and thus of FP, despite polarization-dependent cross-talk in the overall assay.
It is possible to use the invention to measure and correct for the relative exposure times or lamp fluctuations associated with each measurement in the raw data set, if desired. Through these aspects of the invention and others described in more detail below, a multiple-probe FP is achieved, with accurate quantification of FP for each probe despite spectral cross-talk, instrumental polarization sensitivities, and fluctuations in exposure or lamp brightness.
The various features of novelty which characterize the invention are pointed out with particularity in the claims annexed to and forming a part of the disclosure. For a better understanding of the invention, its operating advantages, and specific objects attained by its use, reference should be had to the drawings and descriptive matter in which there are illustrated and described preferred embodiments of the invention.